Stop-flow reversed phase liquid chromatography × size-exclusion chromatography for separation of peptides

Size-exclusion chromatography (SEC) with wide application in peptide analysis presents challenges in determination of molecular weight distribution due to the relatively low resolution. In this study, a stop-flow reversed phase liquid chromatography (RPLC) × SEC system was constructed, aiming at improving the peptide separation in SEC. As the chromatographic dispersion during stop-flow operation might contribute to the band broadening in the first dimension (1st D) RPLC, the effects of different stop-flow operational parameters on the additional band broadening were quantitatively evaluated. Unlike analytes of large molecular size or long retention time with low effective diffusion coefficient (D_eff), additional band broadening was evidently observed for analytes of small molecular size and short retention time (high D_eff). Therefore, optimal flow rate, low column temperature and short stop-flow time were suggested for analyzing small molecules of short retention time. The established stop-flow two-dimensional liquid chromatography (2D-LC) was further tested on protein hydrolysates. The resolution was evidently improved for both heart-cutting and comprehensive 2D-LC analysis (despite additional band broadening in RPLC). Compared with heart-cutting analysis with higher 1st D resolution for selective fractions, comprehensive analysis could provide more complete information about the molecular weight distribution of the eluting solutes along RPLC.